Modification at HuR(S242) alters HuR localization and proliferative influence.
HuR is predominantly nuclear but following exposure to stress and mitogens, it can translocate to the cytoplasm where it stabilizes target mRNAs and/or modulates their translation. Several phosphorylation sites in a central 'hinge" region of HuR have been reported to affect its nucleocytoplasmic shuttle: phosphorylation by PKC at serine (S)221 and by Cdk1 at S202. Here, we investigated if there are additional putative phosphorylation sites within the HuR hinge region capable of influencing its cytoplasmic abundance. We systematically mutated all seven serine residues within the shuttling hinge domain to the nonphosphorylatable residue alanine (A), S197A, S202A, S221A, S229A, S232A, S241A and S242A. Using HeLa cells as the study system, we found that the HuR(S242A) mutant was more abundant in the cytoplasm in both untreated cells and in cells treated with short-wavelength ultraviolet light or with an inhibitor of Cdk1. Conversely, mutation of S242 to aspartic acid (D), rendered the phosphomimetic HuR(S242D) nuclear under all treatment conditions. S242 mutations did not influence HuR stability, but HuR(S242A) showed increased association with target cyclin A2 and cyclin B1 mRNAs. Accordingly, expression of HuR(S242A) led to increased cyclin mRNA stability and heightened cell proliferation rates. Our findings suggest that HuR phosphorylation at S242 hinders its cytoplasmic localization, its function as a posttranscriptional regulator, and its proliferative influence.
Kim
Hyeon Ho
HH
Laboratory of Cellular and Molecular Biology, National Institute on Aging-IRP, National Institutes of Health, Baltimore, Maryland 21224, USA.
Yang
Xiaoling
X
Kuwano
Yuki
Y
Gorospe
Myriam
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D049109:Cell Proliferation; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D055550:Protein Stability; D021381:Protein Transport; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics*
D016213:Cyclins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D050505:Mutant Proteins; D012333:RNA, Messenger; D012694:Serine